The principal goal of this proposal is to identify the epigenetic and morphological effects of lead (Pb) exposure on the neural and neuronal differentiation of human embryonic stem cells (hESCs). Lead poisoning remains a significant health problem in the United States. Nationally, about 250,000 children aged 1-5 years have blood lead levels greater than 10 g of lead per deciliter of blood (current CDC action level). It s known that Pb exposure causes neurological problems in children, such as attention deficit disorder and loss of fine-motor coordination, but the mechanisms of Pb-induced neurotoxicity and the epigenetic basis for how Pb causes neurological deficits is not known. In humans, the brain starts forming at about three weeks after conception and continues to develop and mature well into the first years of life. Early brain development is a very sensitive time during which exposure to toxicants such as Pb can affect further brain development and maturation. In vitro differentiation of embryonic stem cells into neural precursor cells (NPCs) recapitulates the early stages of brain development in humans, giving one the unique opportunity to understand the effects of Pb on the formation of the human brain. The significance of the experiments proposed here is that they will allow one to get a unique look at the impact of Pb on the differentiation an the maturation of NPCs and at the potential changes in the global DNA methylation patterns associated with Pb exposure. These differences will reveal novel mechanisms of Pb neurotoxicity and will also establish a tentative epigenetic outline that can be used as an index for Pb exposure. NPC-derived neurons will be used to understand even subtle changes in genomic DNA CpG methylation status leading to the neurological alterations induced by Pb. Preliminary studies with NPCs derived from hESCs indicate that Pb is an epigenetic disruptor and causes rapid (within 24 hours) reprogramming of global DNA methylation levels. The hypothesis is that Pb causes changes in DNA methylation at specific CpG sites and that hESCs and NPCs will have changes in epigenetic signature that correlate with morphological, molecular, and functional changes in the hESC-derived neural precursors and neurons. Three aims are proposed: Aim 1. Determine the effects of chronic Pb exposure on the epigenetic profile of hESC- derived NPCs. Aim 2. Examine the influence that Pb-mediated DNA methylation changes in candidate genes have on neuronal differentiation and gene expression. Outcomes: This project will allow one to understand how exposure to Pb leads to neural impairment and how the architecture of a DNA methylome leads to the neurotoxic effects of Pb. It will be a prototype study for the application of the emerging field of toxicogenomics to human brain development, where the concepts could be used to devise in vitro assays for a variety of teratogenic toxic agents for other types of cell lineages as well.